Phosphorylation of proteins is a fundamental mechanism for regulating diverse cellular processes. While the majority of protein phosphorylation occurs at serine and threonine residues, phosphorylation at tyrosine residues is attracting a great deal of interest since the discovery that many oncogene products and growth factor receptors possess intrinsic protein tyrosine kinase activity. The importance of protein tyrosine phosphorylation in growth factor signal transduction, cell cycle progression and neoplastic transformation is now well established (Hunter et al., Ann. Rev. Biochem. 54:987–930 (1985), Ullrich et al., Cell 61:203–212 (1990), Nurse, Nature 344:503–508 (1990), Cantley et al, Cell 64:281–302 (1991)).
Biochemical studies have shown that phosphorylation on tyrosine residues of a variety of cellular proteins is a dynamic process involving competing phosphorylation and dephosphorylation reactions. The regulation of protein tyrosine phosphorylation is mediated by the reciprocal actions of protein tyrosine kinases (PTKases) and protein tyrosine phosphatases (PTPases). The tyrosine phosphorylation reactions are catalyzed by PTKases. Tyrosine phosphorylated proteins can be specifically dephosphorylated through the action of PTPases. The level of protein tyrosine phosphorylation of intracellular substances is determined by the balance of PTKase and PTPase activities. (Hunter, T., Cell 58:1013–1016 (1989)).
The protein tyrosine kinases (PTKases) are a large family of proteins that includes many growth factor receptors and potential oncogenes. (Hanks et al., Science 241:42–52 (1988)). Many PTKases have been linked to initial signals required for induction of the cell cycle (Weaver et al., Mol. Cell. Biol. 11, 9:4415–4422 (1991)). PTKases comprise a discrete family of enzymes having common ancestry with, but major differences from, serine/threonine-specific protein kinases (Hanks et al., supra). The mechanisms leading to changes in activity of PTKases are best understood in the case of receptor-type PTKases having a transmembrane topology (Ullrich et al. (1990) supra). The binding of specific ligands to the extracellular domain of members of receptor-type PTKases is thought to induce their oligomerization leading to an increase in tyrosine kinase activity and activation of the signal transduction pathways (Ullrich et al., (1990) supra). Deregulation of kinase activity through mutation or overexpression is a well established mechanism for cell transformation (Hunter et al., (1985) supra; Ullrich et al., (1990) supra).
The protein phosphatases are composed of at least two separate and distinct families (Hunter, T. (1989) supra) the protein serine/threonine phosphatases and the protein tyrosine phosphatases (PTPases).
The protein tyrosine phosphatases (PTPases) are a family of proteins that have been classified into two subgroups. The first subgroup is made up of the low molecular weight, intracellular enzymes that contain a single conserved catalytic phosphatase domain. All known intracellular type PTPases contain a single conserved catalytic phosphatase domain. Examples of the first group of PTPases include (1) placental PTPase 1B (Charbonneau et al., Proc. Natl. Acad. Sci. USA 86:5252–5256 (1989); Chernoff et al., Proc. Natl. Acad. Sci. USA 87:2735–2789 (1989)), (2) T-cell PTPase (Cool et al., Proc. Natl. Acad. Sci. USA 86:5257–5261 (1989)), (3) rat brain PTPase (Guan et al., Proc. Natl. Acad. Sci. USA 87:1501–1502 (1990)), (4) neuronal phosphatase (STEP) (Lombroso et al., Proc. Natl. Acad. Sci. USA 88:7242–7246 (1991)), and (5) cytoplasmic phosphatases that contain a region of homology to cytoskeletal proteins (Gu et al., Proc. Natl. Acad. Sci. USA 88:5867–57871 (1991); Yang et al., Proc. Natl. Acad. Sci. USA 88:5949–5953 (1991)).
Enzymes of this class are characterized by an active site motif of CX5R. Within the motif the Cysteine sulfur acts as a nucleophile which cleaves the P—O bond and releases the phosphate; the Arginine interacts with the phosphate and facilitates nucleophilic attack. In many cases the Cysteine is preceded by a Histidine and the Arginine is followed by a Serine or Threonine. In addition, an Aspartate residue located 20 or more amino acids N terminal to the Cysteine acts as a general acid during cleavage [Fauman, 1996].
The second subgroup of protein tyrosine phosphatases is made up of the high molecular weight, receptor-linked PTPases, termed R-PTPases. R-PTPases consist of a) an intracellular catalytic region, b) a single transmembrane segment, and c) a putative ligand-binding extracellular domain (Gebbink et al., supra).
The structures and sizes of the c) putative ligand-binding extracellular “receptor” domains of R-PTPases are quite divergent. In contrast, the a) intracellular catalytic regions of R-PTPases are highly homologous. All RPTPases have two tandemly duplicated catalytic phosphatase homology domains, with the prominent exception of an R-PTPase termed HPTP.beta., which has “only one catalytic phosphatase domain. (Tsai et al., J. Biol. Chem. 266(16):10534–10543 (1991)).
One example of R-PTPases are the leukocyte common antigens (LCA) (Ralph, S. J., EMBO J. 6:1251–1257 (1987)). LCA is a family of high molecular weight glycoproteins expressed on the surface of all leukocytes and their hemopoietic progenitors (Thomas, Ann. Rev. Immunol. 7:339–369 (1989)). A remarkable degree of similarity is detected with the sequence of LCA from several species (Charbonneau et al., Proc. Natl. Acad. Sci. USA 85:7182–7186 (1988)). LCA is referred to in the literature by different names, including T200 (Trowbridge et al., Eur. J. Immunol. 6:557–562 (1962)), B220 for the B cell form (Coffman et al., Nature 289:681–683 (1981)), the mouse allotypic marker Ly-5 (Komuro et al., Immunogenetics 1:452–456 (1975)), and more recently CD45 (Cobbold et al., Leucocyte Typing III, ed. A. J. McMichael et al., pp. 788–803 (1987)).
Several studies suggest that CD45 plays a critical role in T cell activation. These studies are reviewed in Weiss A., Ann. Rev. Genet. 25:487–510 (1991). In one study, T-cell clones that were mutagenized by NSG and selected for their failure to express CD45 had impaired responses to T-cell receptor stimuli (Weaver et al., (1991) supra). These T-cell clones were functionally defective in their responses to signals transmitted through the T cell antigen receptor, including cytolysis of appropriate targets, proliferation, and lymphokine production (Weaver et al., (1991) supra).
Other studies indicate that the PTPase activity of CD45 plays a role in the activation of pp56.sup.lck, a lymphocyte-specific PTKase (Mustelin et al., Proc. Natl. Acad. Sci. USA 86:6302–6306 (1989); Ostergaard et al., Proc. Natl. Acad. Sci. USA 86:8959–8963 (1989)). These authors hypothesized that the phosphatase activity of CD45 activates pp56.sup.lck by dephosphorylation of a C-terminal tyrosine residue, which may, in turn, be related to T-cell activation.
Another example of R-PTPases is the leukocyte common antigen related molecule (LAR) (Streuli et al., J. Exp. Med. 168:1523–1530 (1988)). LAR was initially identified as a homologue of LCA (Streuli et al., supra). Although the a) intracellular catalytic region of the LAR molecule contains two catalytic phosphatase homology domains (domain I and domain II), mutational analyses suggest that only domain I has catalytic phosphatase activity, whereas domain II is enzymatically inactive (Streuli et al., EMBO J. 9(8):2399–2407 (1990)). Chemically induced LAR mutants having tyrosine at amino acid position 1379 changed to a phenylalanine are temperature-sensitive (Tsai et al., J. Biol. Chem. 266(16):10534–10543 (1991)).
A new mouse R-PTP, designated mRPTP.mu., has been cloned which has a) an extracellular domain that shares some structural motifs with LAR. (Gebbink et al., (1991) supra). In addition, these authors have cloned the human homologue of RPTP.mu. and localized the gene on human chromosome 18.
Two Drosophila PTPases, termed DLAR and DPTP, have been predicted based on the sequences of cDNA clones (Streuli et al., Proc. Natl. Acad. Sci. USA 35 86:8698–8702 (1989)). cDNAs coding for another Drosophila R-PTPase, termed DPTP 99A, have been cloned and characterized (Hariharan et al., Proc. Natl. Acad. Sci. USA 88:11266–11270(1991)).
Other examples of R-PTPases include R-PTPase-.alpha., .beta., .gamma., and .zeta. (Krueger et al., EMBO J. 9:3241–3252 (1990), Sap et al., Proc. Natl. Acad. Sci. USA 87:6112–6116 (1990), Kaplan et al., Proc. Natl. Acad. Sci. USA 87:7000–7004 (1990), Jirik et al., FEBS Lett. 273:239–242 (1990); Mathews et al., Proc. Natl. Acad. Sci. USA 87:4444–4448 (1990), Ohagi et al., Nucl. Acids Res. 18:7159 (1990)). Published application W092/01050 discloses human R-PTPase-.alpha., .beta. and .gamma., and reports on the nature of the structural homologies found among the conserved domains of these three R-PTPases and other members of this protein family. The murine R-PTPase-.alpha. has 794 amino acids, whereas the human R-PTPase-.alpha. has 802 amino acids. R-PTPase-.alpha. has an intracellular domain homologous to the catalytic domains of other tyrosine phosphatases. The 142 amino acid extracellular domain (including signal peptide of RPTPase-.alpha.) has a high serine and threonine content (32%) and 8 potential N-glycosylation sites. cDNA clones have been produced that code for the R-PTPase-.alpha., and R-PTPase-.alpha. has been expressed from eukaryotic hosts. Northern analysis has been used to identify the natural expression of R-PTPase-.alpha. in various cells and tissues. A polyclonal antibody to R-PTPase-.alpha. has been produced by immunization with a synthetic peptide of R-PTPase-.alpha., which identifies a 130 kDa protein in cells transfected with a cDNA clone encoding a portion of R-PTPase-.alpha.
Another example of R-PTPases is HePTP. (Jirik et al, FASEB J. 4:82082 (1990) Abstract 2253). Jirik et al. screened a cDNA library derived from a hepatoblastoma cell line, HepG2, with a probe encoding the two PTPase domains of LCA, and discovered a cDNA clone encoding a new RPTPase, named HePTP. The HePTP gene appeared to be expressed in a variety of human and murine cell lines and tissues.
Since the initial purification, sequencing, and cloning of a PTPase, additional potential PTPases have been identified at a rapid pace. The number of different PTPases that have been identified is increasing steadily, leading to speculations that this family may be as large as the PTKase family (Hunter (1989) supra).
Conserved amino acid sequences in the catalytic domains of known PTPases have been identified and defined (Krueger et al., EMBO J. 9:3241–3252 (1990) and Yi et al., Mol. Cell. Biol. 12:836–846 (1992), which are incorporated herein by reference.) These amino acid sequences are designated “consensus sequences” herein.
Yi et al. aligned the catalytic phosphatase domain sequences of the following PTPases: LCA, PTP1B, TCPTP, LAR, DLAR, and HPTP.alpha., HPTP.beta., and HPTP.gamma. This alignment includes the following “consensus sequences” (Yi et al., supra, FIG. 2(A), lines 1 and 2): DYINAS/N (SEQ ID NO:77), CXXYWP (SEQ ID NO:78), and I/VVMXXXXE (SEQ ID NO:79).
Krueger et al., aligned the catalytic phosphatase domain sequences of PTP1B, TCPTP, LAR, LCA, HPTP.alpha., .beta., .gamma., .GAMMA., .delta., .epsilon. and .zeta. and DLAR and DPTP. This alignment includes the following “consensus sequences: (Krueger et al., supra, FIG. 7, lines 1 and 2): D/NYINAS/N (SEQ ID NO:80), CXXYWP (SEQ ID NO:81), and I/VVMXXXXE (SEQ ID NO:82).
It is becoming clear that dephosphorylation of tyrosine residues can by itself function as an important regulatory mechanism. Dephosphorylation of a C-terminal tyrosine residue has been shown to activate tyrosine kinase activity in the case of the src family of tyrosine kinases (Hunter, T. Cell 49:1–4 (1987)). Tyrosine dephosphorylation has been suggested to be an obligatory step in the mitotic activation of the maturation-promoting factor (MPF) kinase (Morla et al., Cell 58:193–203 (1989)). These observations point out the need in the art for understanding the mechanisms that regulate tyrosine phosphatase activity.
Modulators (inhibitors or activators) of human phosphatase expression or activity could be used to treat a subject with a disorder characterized by aberrant phosphatase expression or activity or by decreased phosphorylation of a phosphatase substrate protein. Examples of such disorders include but are not limited to: an immune, anti-proliferative, proliferative (e.g. cancer), metabolic (e.g. diabetes or obesity), bone (e.g., osteoporosis), neural, and/or cardiovascular diseases and/or disorders, in addition to, viral pathogenesis.
It is clear that further analysis of structure-function relationships among PTPases are needed to gain important understanding of the mechanisms of signal transduction, cell cycle progression and cell growth, and neoplastic transformation.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells, in addition to their use in the production of human phosphatase polypeptides or peptides using recombinant techniques. Synthetic methods for producing the polypeptides and polynucleotides of the present invention are provided. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the human phosphatase polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders, and/or conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides.